Conventionally, in fabrication of a tissue specimen for pathological diagnosis, a paraffin block is fabricated via steps of fixation, dehydration, de greasing, intermediate treatment, impregnation treatment with paraffin and the like, and the paraffin block is sliced, finally, stained by various staining methods, and covered with a cover glass to give a praeparate (prepared specimen) which is the observed by a microscope for pathological diagnosis.
Recently, speed up of the pathological diagnosis is required, and there are developed apparatuses for treatment of various tissue specimens in treatment steps for speed up of the treatment. Also for treatment apparatuses of conducting from a fixing treatment step to a paraffin impregnating treatment step, there are measures of shortening the treatment time, such as provision of pressurizing and pressure reducing mechanisms and a stirring function.
Nowadays, for speed up of the treatment, tissue piece treating apparatuses utilizing microwave energy as described in U.S. Pat. Nos. 4,656,047, 4,839,194 and 5,244,787 are also developed. However, since these technologies use the same design as for microwave ovens used for home cooking, these are utterly different from the technology applying an ultrasonic wave as in the present invention.
An invention according to U.S. Pat. No. 5,089,288 (hereinafter, referred to as “latest prior art”) discloses an invention regarding a method of impregnation of a tissue piece with a paraffin. FIG. 1 shows a schematic sectional view of an impregnation treating bath used in the latest, prior art (FIG. 1 in U.S. Pat. No. 5,089,288).
Matters and drawings described in U.S. Pat. No. 5,089,288 can be derived directly and unambiguously in the Description of the present application.
In the schematic sectional view, (1) represents a treating bath, (2) represents a bath inside, (3) represents a tissue piece, (4) represents a cassette, (5) represents a constant temperature bath, (6) represents an ultrasonic vibrator, (7) represents a cover, (8) represent a liquid surface, (9) represents a connector, (10) represents a pipeline, (11) represents a filter, (12) represents a vacuum pump, (13to 17) represent tanks, and (L) represents liquid level. The treatment and paraffin impregnation treatment of the tissue piece (3) are all conducted in the bath inside (2) of the treating bath (1).
[Basic Structure of Treating Apparatus]
In the treating bath (1), the bath inside (2) and a drug solution flown into the bath inside (2) can be maintained at a previously-set temperature by being over-coated by the constant temperature bath (5).
The constant temperature bath (5) is capable of performing heating, cooling, temperature regulation and temperature control of the bath inside (2) and drug solution with a heat medium (e.g., air and water) which has been appropriately heated, cooled, temperature-regulated or temperature-controlled.
The ultrasonic vibrator (6) installed to the treating bath (1) is capable of oscillating an ultrasonic wave having a wavelength of, for example, 35 to 50 kHz.
The output of the ultrasonic vibrator (6) is 120 watts (when the treating bath is small) to 1200 watts (when the treating bath is large), and can be varied in the range of 100 to several hundreds of watts. The ultrasonic vibrator (6) gives ultrasonic wave energy suitable for the tissue piece (3) and drug solution introduced into the bath inside (2).
The cover (7) on the upper side can perform air tight or vacuum sealing of the treating bath (1).
Each drug solution is introduced up to the liquid level of the bath inside (2) so as to keep a space between the cover (7) and the liquid surface. The connector (9) is installed at a position distant sufficiently from the liquid surface (8) and communicates with the pipeline (10) and the filter (11). The bath inside (2) sealed with the cover (7) can be pressure-reduced or evacuated by a vacuum pump (12).
[Structure of Cassette]
The cassette (4) is a box-shaped vessel having a netted bottom part and a netted cover, generally used for a paraffin-impregnation treatment or paraffin-embedding of a tissue piece (3). A drug solution flown into the bath inside (2) of the treating bath (1) enters through a mesh of the net of the bottom part and the cover and comes into contact with the tissue piece (3), thus, the tissue piece (3) can be treated.
[Constitution of Tank]
The tanks (13to 17) are connected to the treating bath. The tank (13) stores a fixing agent (e.g., isopropyl alcohol) for the tissue piece (3), the tank (14) stores a first dehydrating treatment liquid (70 wt % isopropyl alcohol/30 wt % water solution) used for a partial dehydrating treatment, the tank (15) stores a second dehydrating treatment liquid (100 wt % isopropyl alcohol) used for an intermediate dehydrating treatment, the tank (16) stores a third dehydrating treatment liquid (100 wt % isopropyl alcohol) used for a complete dehydrating treatment, and the tank (17) stores a molten paraffin. Drug solutions in the tanks are maintained at respective previously-set temperatures. The set temperature of the tank (17) is, for example, a temperature (e.g., 58° C.) at which a paraffin can be kept at liquid state. The set temperatures of the tanks (13 to 16) are, for example, a temperature (e.g., 58° C.) lower than the boiling point (82.8° C.) of isopropyl alcohol under standard atmospheric pressure. The drug solution stored in the tanks (13 to 17) can be introduced into the bath inside (2) of the treating bath (1) or the drug solution can be returned to the tanks (13 to 17) from the bath inside (2) by a drug solution feeding system (not shown) composed of a pump, valve and pipeline.
[Summary of Treating Procedure of Tissue Piece]
The tissue piece (3) is subjected to a fixing treatment with a fixing agent in the tank (13), then, subjected to a partial dehydrating treatment with a first dehydrating treatment liquid in the tank (14), then, subjected to an intermediate dehydrating treatment with a second dehydrating treatment liquid in the tank (15), then, subjected to a complete dehydrating treatment with a third dehydrating treatment liquid in the tank (16), then, subjected to a paraffin impregnating treatment with a molten paraffin in the tank (17). By adopting such an operation procedure, it becomes possible to carry out a fixing treatment, dehydrating treatment and paraffin impregnating treatment of a tissue piece without necessity of conducting an intermediate treatment with an intermediate agent (treatment conducted between dehydrating and degreasing treatments and a paraffin impregnating treatment).
[Fixing Treatment of Tissue Piece]
A cassette (4) in which a tissue piece (3) is set is set in the bath inside (2), then, the bath inside (2) is sealed with the cover (7). After sealing, a fixing agent (e.g., isopropyl alcohol) is introduced from the tank (13) up to the liquid level (L) of the bath inside (2). The temperature of the fixing agent is raised, by the ultrasonic wave energy of the ultrasonic vibrator (6), up to a temperature (for example, 58° C.) at which a paraffin can be maintained in liquid state. After completion of the fixing treatment of the tissue piece (3), the fixing agent is returned to the tank (13).
[First Dehydrating Treatment of Tissue Piece]
After completion of the fixing treatment, a first dehydrating treatment liquid (70 vol % isopropyl alcohol/30 vol % water solution) is introduced from the tank (14) up to the liquid level (L) of the bath inside (2). Since treatment liquids prepared in the tanks (14 to 17) are maintained previously at a treatment temperature (for example, 58° C.), the isopropyl alcohol introduced is maintained already at a treatment temperature (for example, 58° C.). Partial dehydration of a tissue piece carried out by soaking into 70 vol % isopropyl alcohol is promoted by irradiation of the first dehydrating treatment liquid and the tissue piece (3) with an ultrasonic wave. After completion of the first dehydrating treatment, the first dehydrating treatment liquid is returned to the tank (14).
[Second Dehydrating Treatment of Tissue Piece]
After completion of the first dehydrating treatment, a second dehydrating treatment liquid (100 vol % isopropyl alcohol) is introduced from the tank (15) up to the liquid level (L) of the bath inside (2). Further dehydration and degreasing of the tissue piece (3) are promoted by irradiation of the second dehydrating treatment liquid and the tissue piece (3) with an ultrasonic wave. After completion of the second dehydrating treatment, the second dehydrating treatment liquid is returned to the tank (15).
[Third Complete Dehydrating Treatment of Tissue Piece]
After completion of the second dehydrating treatment, a third dehydrating treatment liquid (100 vol % isopropyl alcohol) is introduced from the tank (16) up to the liquid level (L) of the bath inside (2). Complete dehydration and degreasing of the tissue piece (3) are promoted by irradiation of the third dehydrating treatment liquid and the tissue piece (3) with an ultrasonic wave. After completion of the third dehydrating treatment, the third dehydrating treatment liquid is returned to the tank (16).
[Paraffin Impregnating Treatment of Tissue Piece]
After completion of the third dehydrating treatment, a molten paraffin is introduced from the tank (17) up to the liquid level (L) of the bath inside (2), and after the introduction, the treating bath (1) is sealed with the cover (7). The temperature of the molten paraffin is maintained at a temperature capable of keeping the liquid state of the paraffin, for example, at 58° C. After sealing of the treating bath (1) with the cover (7), the pressure of the bath inside (2) is reduced to a degree of vacuum of the order of 10 kPa by a vacuum pump (12).
It is desirable to perform this pressure reduction as quickly as possible, however, by irradiation with an ultrasonic wave during pressure reduction, the temperature of isopropyl alcohol in the bath inside (2) reaches the boiling point to promote evaporation thereof, thereby increasing the pressure reduction speed. Impregnation of the molten paraffin into the tissue piece (3) is promoted by irradiating the tissue piece (3) and the molten paraffin with an ultrasonic wave while maintaining the degree of vacuum of the bath inside (2). By performing pressure reduction of the bath inside (2) in the treating bath (1), the boiling point of isopropyl alcohol (the boiling point under standard atmospheric pressure is 82.8° C.) yet remaining in the tissue piece (3) lowers.
Since the boiling point of isopropyl alcohol under vacuum is lower than the treatment temperature (58° C. at which a paraffin is capable of maintaining liquid state), isopropyl alcohol vaporizes out of the tissue piece (3) under vacuum and passes through without being dissolved in the molten paraffin around the tissue piece (3), and is discharged via a connector (9). Therefore, the molten paraffin is not polluted with isopropyl alcohol. A void generated by evaporation of isopropyl alcohol out of the tissue piece (3) is substituted immediate with the molten paraffin.
Since the paraffin impregnating treatment can be carried out with leaving utterly no air bubble in the tissue piece (3), even a tissue containing air such as a lung tissue can be completely substituted with a paraffin. By such a process, it becomes possible to conduct the paraffin impregnating treatment and paraffin embedding of tissue piece (3) in an extremely short period of time. In the case of a very thin tissue piece having a thickness of 2 mm, the treatment is possible in only 20 to 30 minutes, and in the case of a thick tissue piece having a thickness of 3 to 4 mm, the treatment is possible in 1 to 1.5 hours. Irrespective of very short treatment time, the paraffin impregnating treatment and paraffin embedding can be carried out with extremely high quality, thus, a sliced piece suitable for a histological microscopic check can be provided.
[Recovery of Liquid Paraffin]
After completion of the paraffin impregnating treatment of a tissue piece, the molten paraffin is returned to the tank (17) from the bath inside (2). Since the molten paraffin is not polluted by isopropyl alcohol, there is no need of a special pollution check for the recovered isopropyl alcohol and the tank (17). It may be advantageous to add the paraffin to the tank in an amount corresponding to the use amount of the paraffin impregnated into the tissue piece (3), thus, this procedure is very economical.
[Fabrication of Paraffin Block of Tissue Piece]
After the paraffin impregnating treatment of the tissue piece (3), a paraffin block is fabricated by a usual method.
[Merit (1) in Latest Prior Art]
As a first merit in the latest prior art, there is no necessity of conducting an intermediate treatment with an intermediate agent (treatment conducted between dehydrating and degreasing treatments and a paraffin impregnating treatment), and in the case of a very thin tissue piece having a thickness of 2 mm, the treatment is possible in only 20 to 30 minutes, and in the case of a thick tissue piece having a thickness of 3 to 4 mm, the treatment is possible in 1 to 1.5 hours. Irrespective of very short treatment time, the paraffin impregnating treatment and paraffin embedding can be carried out with extremely high quality, thus, a sliced piece suitable for a histological microscopic check can be provided.
[Merit (2) in Latest Prior Art]
As a second merit in the latest prior art, reaching of the temperature of isopropyl alcohol in the bath inside to the boiling point and evaporation thereof are promoted to accelerate the pressure reduction speed, by irradiating an ultrasonic wave during pressure reduction in performing reduction of the pressure in the bath inside down to a degree of vacuum of the order of 10 kPa by a vacuum pump after sealing the treating bath with a cover.
[Merit (3) in Latest Prior Art]
As a third merit in the latest prior art, since the paraffin impregnating treatment can be carried out with leaving utterly no air bubble in the tissue piece, even a tissue containing air such as a lung tissue can be completely substituted with a paraffin. By such a process, it becomes possible to conduct the paraffin impregnating treatment and paraffin embedding of tissue piece (3) in an extremely short period of time.
[Merit (4) in Latest Prior Art]
As a fourth merit in the latest prior art, since the molten paraffin is not polluted by isopropyl alcohol, there is no need of a special pollution check for the recovered isopropyl alcohol and the storage tank.
[Merit (5) in Latest Prior Art]
As a fifth merit in the latest prior art, since it may be advantageous to add the paraffin to the tank in an amount corresponding to the use amount of the paraffin impregnated into the tissue piece, this procedure is very economical.
[Problem (1) in Latest Prior Art]
As a first problem in the latest prior art, since the apparatus adopts a constitution of over-coating of the treating bath (1) by a constant temperature bath (5), the constant temperature bath (5) is bulky and has a large occupied volume, thus, usability and the degree of freedom of installation thereof are extremely poor in a narrow laboratory.
[Problem (2) in Latest Prior Art]
As a second problem in the latest prior art, since the apparatus adopts a constitution of over-coating of the treating bath (1) by a constant temperature bath (5), the weight of the apparatus is larger in the case of adoption of water as a heat medium of the constant temperature bath (5).
[Problem (3) in Latest Prior Art]
As a third problem in the latest prior art, since the apparatus adopts a constitution of over-coating of the treating bath (1) by a constant temperature bath (5) using water or air as a heat medium, the accuracy of temperature control of the bath inside (2) in the treating bath (1) is low.
[Problem (4) in Latest Prior Art]
As a fourth problem in the latest prior art, since the apparatus adopts a constitution of over-coating of the treating bath (1) by a constant temperature bath (5) using water or air as a heat medium and indirect heating and cooling are carried out in this constitution, the heat efficiency is low and electric power consumption is large.
[Problem (5) in Latest Prior Art]
As a fifth problem in the latest prior art, since the apparatus adopts a constitution of over-coating of the treating bath (1) by a constant temperature bath (5) using water or air as a heat medium, there are required exchange of the polluted heat medium, exchange of parts (e.g., nichrome wire) of a complicated apparatus, regulation of a thermostat (e.g., bimetal) and the like, thus, the maintenance is complicated and a trouble tends to occur.
Patent Literature 1: U.S. Pat. No. 5,089,288